WP1: We obtained a 6 month extension + ensured deliverables + milestones were achieved in coherence with the updated timelines, extending the GANTT span from 48 to 54 months.
WP2: We submitted a major review on modeling microRNA regulation of the TGF-β/SMAD signaling pathways for seizure control in temporal lobe epilepsy, had one research article on microRNA-based modeling of TGF-β/SMAD signaling for seizure control in temporal lobe epilepsy accepted for publication, published a paper in IEEE Transactions on Molecular, Biological, and Multi-Scale Communications focusing on molecular communication frameworks for brain signal analysis + have 2 additional manuscripts in progress on molecular communication models + loss budget estimation for tsRNA detection in the brain. We integrated AGO2-immunoprecipitated small RNA sequencing with matched transcriptomic and proteomic datasets across seven stages of epileptogenesis in the perforant pathway stimulation (PPS) rat model of temporal lobe epilepsy. This work provides the first integrative multi-omics view of tsRNA activity in epilepsy and highlights novel RNA-mediated mechanisms with potential relevance for disease progression.
WP3: We determined the expression and secretion of GDNF with both a constitutive and a regulated pathway in human ARPE-19 cells and rat rMC-1 cells and also analysed the contribution of intracellular Ca2+ in promoting GDNF secretion. We achieved high expression and secretion of GDNF in engineered ARPE-19 cells that overexpress GDNF alone, and the group also produced lentiviral vectors that will be used to generate cells over-expressing both P2X7 (the ATP-gated purinergic P2X7 receptor) and GDNF (according to the fail-safe strategy). We have established assays for the detection of extracellular 5’-tRFs by quantitative PCR and extracellular GDNF by ELISA, enabling the testing of the engineered cells. 5’tRFs were also quantified in the CSF (cerebrospinal fluid) and serum of epileptic rats, in relation to seizure occurrence and inter-ictal activity.
WP4: We further optimized hybrid scaffolds of collagen hydrogels and laminin-functionalized electrospun polymer nanorods as a biomimetic habitant for engineered ARPE-19 cells mimicking the brain extracellular microenvironment with the implementation of finite element simulations + further introduced innovative design approaches for nano-or micro-fiber tubes via coaxial electrospinning and via additive manufacturing. We successfully developed a sealed porous encapsulating membrane using commercially available polyethersulfone (PES) tubes.We studied the permeability of the PES tubes to naked mRNA, lipid nanoparticle-encapsulated mRNA, exosomes, and GDNF using ELISA. The results confirmed effective bidirectional transport of these biomolecules across the device membrane.
WP5: We measured the release of GDNF from the devices into the local brain environment and studied the effects of this on animal behaviours. We also tested the effects of encapsulated cell devices that release GDNF on seizures in the RCSI mouse model of epilepsy. We completed an extensive gene expression profiling of the RNA landscape in the tissue surrounding the explanted encapsulated device. The findings represent a comprehensive characterisation of the fuctionality of the encapsulated device + the impact of GDNF on brain excitability + the underlying gene expression programmes.
WP6: Dissemination - we successfully promoted the project via visible online platforms including an active website + social media channels. The website shows public deliverables, publications, news + events, reports of communication activities, results, opinions; operating as the supporting pillar of the project’s online image. It was updated during the project’s lifetime providing a permanent reference for PRIME work. D6.4 provides collective data of the impact metrics + statistical figures for key milestones across the project. Exploitation - despite the low TRL of the project a number of exploitable deliverables have already been identified + subjected to a long-term Technology Transfer Strategy. In addition to the IPR Management + Patent Filing in D6.4 we also out a Sustainability Plan.
